Utility of nematode small molecules

ABSTRACT

The present invention relates to methods of treating immune disorders and/or inflammation using certain modulator compounds. In one embodiment, the present invention provides a method of treating an immune and inflammatory disorders disorder by administering a composition comprising a therapeutically effective dosage of an ascaroside compound, or a mixture of ascaroside compounds, or a mixture containing at least one ascaroside.

This application is a §371 national-stage application based on PatentCooperation Treaty Application serial number PCT/US2012/050031, filedAug. 8, 2012, which claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/521,295, filed Aug. 8, 2011, and U.S.Provisional Patent Application Ser. No. 61/620,343, filed on Apr. 4,2012, and U.S. Provisional Application Ser. No. 61/620,331, filed onApr. 4, 2012, and U.S. Provisional Patent Application Ser. No.61/620,348, each of which is hereby incorporated by reference in itsentirety.

GOVERNMENT RIGHTS

This invention was made with United States Government support under NIHAgreement No. GM085285. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to the field of biotechnology, specifically toimmunomodulation and nematodes.

BACKGROUND OF THE INVENTION

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Nematodes are the most abundant animals in the world, found to inhabitsulfurous sediment, deep-sea trenches, human lymph nodes, pigintestines, plant roots, whale placenta, arctic ice, and many otherecosystems, making them one of the most successful groups of animals onearth. Many nematode species are known to parasitize humans; arelationship that is thought to have existed for thousands of years.

The rise of immune disorders in the industrialized world has beenconcomitant with the decline of endemic parasitisim, lending way to theetiologic theory that humans are creating an inappropriate immuneresponse that had previously been focused on overcoming the suppressivemechanisms of parasites. Nematodes have been in use to treat some immunedisorders. There is a need in the art to develop novel compounds andmethods of treating, alleviating, and/or preventing an adverse immuneresponse and/or disorder in the place of live organisms.

SUMMARY OF THE INVENTION

Small molecules produced specifically by nematodes can mimic thepresence of nematodes and thus be used to promote health. Variousembodiments include a method of alleviating, treating, or preventing adisorder in a subject, comprising the provision of a compositioncomprising a compound of the formula:

or a pharmaceutical equivalent, derivative, analog and/or salt thereof,and administering a therapeutically effective dosage of the compositionto the subject. In another embodiment, the disorder is an inflammatoryand/or immune disorder. In another embodiment, R¹ is H, —C(R)₃, —OR,—N(R)₂, halogen, an alkyl, a haloalkyl, an alkenyl, or a haloalkenyl. Inanother embodiment, R^(1′) is absent, H, —C(R)₃, —OR, —N(R)₂, halogen,an alkyl, a haloalkyl, an alkenyl, or a haloalkenyl. In anotherembodiment, R² is a moiety of the formula:

or a pharmaceutical equivalent, derivative, analog and/or salt thereof.In another embodiment, R³ is H, —CR⁶R⁷R⁸, —C(O)R⁸, an alkyl, ahaloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl, aheterocyclyl, a cycloalkyl, a cycloalkenyl, an acyl, an amino acid, anucleoside, a monosaccharide having 5 or 6 carbon atoms, or a bondconnecting to R⁵ of another unit of Formula I. In another embodiment, R⁴is H, —CR⁶R⁷R⁸, —C(O)R⁸, an alkyl, a haloalkyl, an alkenyl, ahaloalkenyl, an aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, acycloalkenyl, an acyl, an amino acid, a nucleoside, a monosaccharidehaving 5 or 6 carbon atoms, or a bond connecting to R⁵ of another unitof Formula I. In another embodiment, R⁵ is H, —OH, —OR⁶, —OCR⁶R⁷R⁸,—CR⁶R⁷R⁸, —NH₂, —NHR⁶, —NR⁶R⁷, halogen, an alkyl, a haloalkyl, analkenyl, a haloalkenyl, an aryl, a heteroaryl, an arylalkyl, aheterocyclyl, a cycloalkyl, a cycloalkenyl, an acyl, an amino acid, anucleoside, a monosaccharide having 5 or 6 carbon atoms, or a bondconnecting to R³ or R⁴ of another unit of Formula I. In anotherembodiment, the disorder is acne vulgaris, asthma, autoimmune diseases,celiac disease, chronic prostatitis, glomerulonephritis,Hypersensitivities, Inflammatory bowel diseases, pelvic inflammatorydisease, reperfusion injury, rheumatoid arthritis, sarcoidosis,transplant rejection, vasculitis, Interstitial cystitis, lupus,scleroderma, certain types of hemolytic anemia, type one diabetes,graves disease, multiple sclerosis, Goodpasture's syndrome, perniciousanemia, some types of myopathy, seasonal allergy, mastocytosis,perennial allergy, anaphylaxis, food allergy, allergic rhinitis, atopicdermatitis, and/or autism. In another embodiment, the disorder isasthma. In another embodiment, the subject is a human. In anotherembodiment, the subject is selected from the group consisting ofprimates, humans, equines, horses, cattle, cows, swine, sheep, rodents,rats, pets, cats, dogs, and guinea pigs. In another embodiment, thecomposition comprises one or more compounds selected from the groupconsisting of ascr#1, ascr#3, ascr#7, ascr#8, ascr#9, and ascr#10.

Other embodiments include a method of reducing inflammation in asubject, comprising providing a composition comprising a compound of theformula:

or a pharmaceutical equivalent, derivative, analog and/or salt thereof,and administering a therapeutically effective dosage of the compositionto the subject. In another embodiment, R¹ is H, —C(R)₃, —OR, —N(R)₂,halogen, an alkyl, a haloalkyl, an alkenyl, or a haloalkenyl. In anotherembodiment, R^(1′) is absent, H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl,a haloalkyl, an alkenyl, or a haloalkenyl. In another embodiment, R² isa moiety of the formula:

or a pharmaceutical equivalent, derivative, analog and/or salt thereof.In another embodiment, R³ is H, —CR⁶R⁷R⁸, —C(O)R⁸, an alkyl, ahaloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl, aheterocyclyl, a cycloalkyl, a cycloalkenyl, an acyl, an amino acid, anucleoside, a monosaccharide having 5 or 6 carbon atoms, or a bondconnecting to R⁵ of another unit of Formula I. In another embodiment, R⁴is H, —CR⁶R⁷R⁸, —C(O)R⁸, an alkyl, a haloalkyl, an alkenyl, ahaloalkenyl, an aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, acycloalkenyl, an acyl, an amino acid, a nucleoside, a monosaccharidehaving 5 or 6 carbon atoms, or a bond connecting to R⁵ of another unitof Formula I. In another embodiment, R⁵ is H, —OH, —OR⁶, —OCR⁶R⁷R⁸,—CR⁶R⁷R⁸, —NH₂, —NHR⁶, —NR⁶R⁷, halogen, an alkyl, a haloalkyl, analkenyl, a haloalkenyl, an aryl, a heteroaryl, an arylalkyl, aheterocyclyl, a cycloalkyl, a cycloalkenyl, an acyl, an amino acid, anucleoside, a monosaccharide having 5 or 6 carbon atoms, or a bondconnecting to R³ or R⁴ of another unit of Formula I. In anotherembodiment, further comprising a reduction in eosinophils in thesubject. In another embodiment, further comprising a reduction in mucusproduction in the subject. In another embodiment, the subject is ahuman. In another embodiment, the subject is selected from the groupconsisting of primates, humans, equines, horses, cattle, cows, swine,sheep, rodents, rats, pets, dogs, and guinea pigs. In anotherembodiment, the composition comprises one or more compounds selectedfrom the group consisting of ascr#1, ascr#3, ascr#7, ascr#8, ascr#9, andascr#10. In another embodiment, the composition comprises an scarosidenaturally produced by a nematode. In another embodiment, the compositioncomprises an ascaroside naturally produced from a Nippostrongylusbrasiliensis. In another embodiment, the inflammation is associated withacne vulgaris, asthma, autoimmune diseases, celiac disease, chronicprostatitis, glomerulonephritis, Hypersensitivities, Inflammatory boweldiseases, pelvic inflammatory disease, reperfusion injury, rheumatoidarthritis, sarcoidosis, transplant rejection, vasculitis, Interstitialcystitis, lupus, scleroderma, certain types of hemolytic anemia, typeone diabetes, graves disease, multiple sclerosis, Goodpasture'ssyndrome, pernicious anemia, some types of myopathy, seasonal allergy,mastocytosis, perennial allergy, anaphylaxis, food allergy, allergicrhinitis, atopic dermatitis, and/or autism.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts, in accordance with an embodiment herein, chemoattractionassay to reveal a gender specific mate finding cue in Panagrellusredivivus. FIG. 1(a) depicts a chemoattraction assay. A transparenttemplate is attached below a 5-cm plate with a 16-mm bacterial lawn todelineate two scoring regions into which 0.64, of the experimentalsample and control sample are placed. Ten P. redivivus males are placedat points equidistant from the two scoring regions and the plate isrecorded for 20 minutes (at a rate of 1 frame per second). FIG. 1(b)depicts attraction is measured using software that computes the ratio ofworm-pixels to total pixels for each scoring region (left panels). Theoutput is a plot of worm occupancy ratio vs. time for both scoringregions (right panel). The shown graph indicates strong attraction of P.redivivus males to the experimental sample.

FIG. 2 depicts, in accordance with an embodiment herein, purification ofthe female sex pheromone in Panagrellus redivivus. FIG. 2(a) depictsPanagrellus redivivus were grown in mixed-gender liquid cultures and thetotal supernatant was fractionated using C18 solid phase extraction, ionexchange, and high performance liquid chromatography. Male attractionwas measured via the Attraction Bioassay (see FIG. 1). FIG. 2(b) depictsthe total worm water was divided using 50% MeOH and 90% MeOH extractionsvia C18 SPE, with significant male attraction to both the combinedfractions (total) and 50% MeOH fraction. FIG. 2(c) depicts the 50% MeOHfraction was subdivided using ion exchange, with no male attraction tothe cation fractions and significant attraction to both the 500 mM and1M anion fractions. FIG. 2(d) depicts the 500 mM and 1M anion fractionswere further subdivided using HPLC. The compound eluted at 10 minutesproduced full male attraction, which was then identified as ascr#1 usingliquid chromatography mass spectrometry (LCMS) and nuclear magneticresonance (NMR). FIG. 2(e) depicts ascr#1 was then synthesized andtested at different doses, demonstrating that males were attracted toascr#1 at concentrations between 102 fmol and 106 fmol whereas femaleswere not attracted to ascr#1 between 100 and 106 fmol. Error bars, S.D.P values were determined using Student's t-test.** P<0.01.

FIG. 3 depicts methods for HPLC-ESI-MS.

FIG. 4 depicts, in accordance with an embodiment herein, thatascarosides are produced by a wide range of nematode species. Resultsfrom the HPLC-MS analysis of worm media samples obtained from incubatingworms for 6 h at 1 worm/μL. Parasitic species indicated as infectivejuveniles (IJ) and adults (designated as “A”) were collected separately;all other samples were obtained from mixed stage cultures. Manynematodes produce species-specific, but partially overlapping blends ofascarosides. Colors in this heatmap represent different relativeabundance of ascarosides in the analyzed species.

FIG. 5 depicts, in accordance with an embodiment herein, thatascarosides mediate behavior of nematode species from diverse generas.Species that were amenable to test in the attraction assay (sufficientmovement, unbiased direction, reproducible controls) were scored forattraction or repulsion toward three concentrations (1 mM, 104, and 1nM: from left to right) of 13 different ascarosides. Pvalues weredetermined using the Student's t-test with a P<0.05. The score “0”represents any findings where P>0.05.

FIG. 6 depicts, in accordance with an embodiment herein, similarassembly of signaling molecules in nematodes and bacteria. N-acylhomoserine lactones (AHLs) play important roles in bacterial quorumsensing and are produced and sensed by many Gramnegative as well as someGram-positive bacteria. All AHLs are based on homoserine lactone andfeature species-specific variations in the N-acyl chain. Ascarosides areassembled in a very similar fashion, based on the dideoxysugarascarylose as invariable scaffold to which a variable lipid chain isattached.

FIG. 7 depicts, in accordance with an embodiment herein, sensitizationwith OVA+Control/Alum (N=3) and sensitization with OVA+NES/Alum (N=3),in Nippostrongylus brasiliensis. The experimental model is ovalbumininducation of asthma. FIG. 7 further depicts lung histology which showsmarked reduction in the inflammation when asthmatic mice were giventotal* hookworm supernatant, as measured by decreased cellularinfiltration and decreased mucus hyper-production. Quantitative mRNAanalysis demonstrates diminished expression of genes associated withmucus production. * Total hookworm water=worms cultured in media, wormsfiltered out. No worm, living or dead, is introduced into the asthmamodel. Furthermore, proteins were eliminated by denaturing at 100 C for25 minutes, and filtering through a 10 kDa filter. FIG. 7 depicts thehistology of mice treated with NES. FIG. 7A shows HE staining of lungsections. The intense infiltration of inflammatory cells, predominantlyeosinophils, into the lungs was observed near the bronchioles andvessels of mice sensitized with Control+OVA. When mice were sensitizedwith NES together with OVA, the cellular infiltration into the lungs wasdramatically reduced. FIGS. 7B and 7C shows PAS (periodic acid-Schiff)staining and the measurement of mRNA expression of Gob5, Muc5a/c andMuc5b in the lung indicated a decrease in mucus hyperproduction in micesensitized with NES together with OVA.

FIG. 8 depicts, in accordance with an embodiment herein, in vitrodifferentiation of Th1/Th2 cells. The effect of NES on thedifferentiation of Th1/Th2 cells is examined. There does not seem to bea direct effect of NES on the differentiation of Th1/Th2 in terms ofIL-4/IFNg production.

FIG. 9 depicts, in accordance with an embodiment herein, in vitrodifferentiation of Th1/Th2 cells. The effect of the antibiotic isexamined, where NES appears to be effective with antibiotics and doesnot negatively disrupt the mouse gut.

FIG. 10 depicts, in accordance with an embodiment herein, synthesizedascrs demonstrates reduction in eosinophils. The effects of ascarosidesare examined for an allergic airway inflammation. It appears thatespecially Ascr#7 has an inhibitory effect for allergic airwayinflammation. The inventors demonstrated that ascr#1, 3, 7, and 10 wasproduced in Nippostrongylus brasiliensis, and then tested synthesizedcompounds individually. The reduction of eosinophils are markedlyreduced when given very small amounts of ascr#7 (purple), comparable tothe effect of total hookworm supernatant (black bar). Ascr#1 and ascr#3show some effect as well.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley& Sons (New York, N.Y. 2001); and Sambrook and Russel, MolecularCloning: A Laboratory Manual 3rd ed., Cold Spring Harbor LaboratoryPress (Cold Spring Harbor, N.Y. 2001), provide one skilled in the artwith a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

As used herein, the following terms, unless otherwise indicated, shallbe understood to have the following meanings. If not defined otherwiseherein, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of ordinary skill in the art towhich this invention belongs. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

The term “alkyl” refers to an aliphatic hydrocarbon group which may be alinear, branched, or cyclic hydrocarbon structure or combinationthereof. Representative alkyl groups are those having 24 or fewer carbonatoms, for instance, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, i-pentyl, n-hexyl, and the like. Lower alkylrefers to alkyl groups having about 1 to about 6 carbon atoms in thechain. Branched alkyl means that one or more lower alkyl groups such asmethyl, ethyl, or propyl are attached to a linear alkyl chain.

The statement that alkyl is intended to include linear, branched, orcyclic hydrocarbon structures and combinations thereof means that an“alkyl” group also includes the following combination of linear andcyclic structural elements

(and similar combinations).

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Branched alkenyl means that one or more lower alkylgroups such as methyl, ethyl, or propyl are attached to a linear alkenylchain. Representative straight chain and branched alkenyls are thosehaving about 2 to about 6 carbon atoms in the chain, for instance,ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl,2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

The term “halogen” refers to fluoro, chloro, bromo, and iodo.

The term “haloalkyl” refers to a branched or straight-chain alkyl asdescribed above, substituted with one or more halogens.

The term “haloalkenyl” refers to a branched or straight-chain alkenyl asdescribed above, substituted with one or more halogens.

The term “aryl” means an aromatic monocyclic or multi-cyclic(polycyclic) ring system of 6 to about 19 carbon atoms, for instance,about 6 to about 10 carbon atoms, and includes arylalkyl groups.Representative aryl groups include, but are not limited to, groups suchas phenyl, naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl,pyrenyl, triphenylenyl, chrysenyl, and naphthacenyl.

The term “arylalkyl” means an alkyl residue attached to an aryl ring.Examples are benzyl, phenethyl, and the like.

The term “heteroaryl” means an aromatic monocyclic or multi-cyclic ringsystem of about 5 to about 19 ring atoms, for instance, about 5 to about10 ring atoms, in which one or more of the atoms in the ring systemis/are element(s) other than carbon, for example, nitrogen, oxygen,and/or sulfur. As is well known to those skilled in the art, heteroarylrings have less aromatic character than their all-carbon counter parts.Thus, for the purposes of the invention, a “heteroaryl” group need onlyhave some degree of aromatic character. For instance, in the case ofmulti-cyclic ring systems, only one of the rings needs to be aromaticfor the ring system to be defined as “heteroaryl”. Exemplary heteroarylscontain about 5 to 6 ring atoms. The prefix aza, oxa, thia, or thiobefore heteroaryl means that at least a nitrogen, oxygen, or sulfuratom, respectively, is present as a ring atom. A nitrogen, carbon, orsulfur atom in the heteroaryl ring may be optionally oxidized; thenitrogen may optionally be quaternized. Representative heteroarylsinclude, but are not limited to, purinyl, pyridyl, 2-oxo-pyridinyl,pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, furanyl, pyrrolyl,thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, indolyl,isoindolyl, benzofuranyl, benzothiophenyl, indolinyl, 2-oxoindolinyl,dihydrobenzofuranyl, dihydrobenzothiophenyl, indazolyl, benzimidazolyl,benzooxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl,benzotriazolyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl,pthalazinyl, quinoxalinyl, and the like.

The terms “cycloalkyl” and “cycloalkenyl” refer to a non-aromatic,saturated (cycloalkyl) or unsaturated (cycloalkenyl), mono- ormulti-cyclic ring system of about 3 to about 8 carbon atoms, forinstance, about 5 to about 7 carbon atoms. Exemplary cycloalkyl andcycloalkenyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclophenyl,anti-bicyclopropane, syn-tricyclopropane, and the like.

As used herein, “heterocycle” or “heterocyclyl” refers to a stable 3- to18-membered ring (radical) which is saturated, unsaturated, or aromatic,and which consists of carbon atoms and from one to five heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur. Forpurposes of this invention, the heterocycle may be a monocyclic,bicyclic, or a polycyclic ring system, which may include fused, bridged,or spiro ring systems, including bicyclic rings in which any of theabove heterocycles are fused to a benzene ring. The nitrogen, carbon, orsulfur atoms in the heterocycle may be optionally oxidized; the nitrogenatom may be optionally quaternized; and the ring may be partially orfully saturated. The heterocycle may be attached via any heteroatom orcarbon atom. Heterocycles include heteroaryls as defined below. Examplesof such heterocycles include, without limitation, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. Furtherheterocycles and heteroaryls are described in Katritzky et al., eds.,Comprehensive Heterocyclic Chemistry: The Structure, Reactions,Synthesis and Use of Heterocyclic Compounds, Vol. 1-8, Pergamon Press,N.Y. (1984), which is hereby incorporated by reference in its entirety.

The term “acyl” refers to groups of from 1 to 8 carbon atoms of astraight, branched, or cyclic configuration, saturated, unsaturated, oraromatic, and combinations thereof, attached to the parent structurethrough a carbonyl functionality. One or more carbons in the acylresidue may be replaced by nitrogen, oxygen, or sulfur as long as thepoint of attachment to the parent remains at the carbonyl. Examplesinclude acetyl (Ac), benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl, and the like.

The term “amino acid” refers to the fragment of an amino acid thatremains following amide bond formation via reaction of the amino acidcarboxyl group with an amino group of another molecule. The amino acidcan be in D- or L-configuration. Suitable amino acids include α-aminoacids, β-amino acids, γ-amino acids, δ-amino acids, and ε-amino acids,and include not only natural amino acids (i.e., those found inbiological systems, including the twenty amino acids found in naturalproteins), but also naturally-occurring variants of such amino acids, aswell as synthetic amino acids and their analogues known to those skilledin the art. Exemplary amino acids include the twenty natural aminoacids, 4-hydroxyproline, hydroxyysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine, and methionine sulfone.

The term “pyrimidine” refers to a heteroaromatic compound containing abenzene ring with two carbon atoms replaced by two nitrogen atoms(diazine). For instance, the following moiety having the carbon atoms atpositions 1 and 3 replaced by nitrogen atoms is considered a pyrimidine:

This term, as it is defined herein, also includes its isomeric forms ofdiazine, such as pyridazine, with the nitrogen atoms in positions 1 and2; and pyrazine, with the nitrogen atoms in positions 1 and 4. The term“pyrimidine” also generally includes its analogues and derivatives. Forinstance, the natural nucleobases, cytosine (C), thymine (T), and uracil(U), are pyrimidine derivatives. The term “purine” refers to aheteroaromatic compound containing a pyrimidine ring fused to animidazole ring. The term “purine” also generally includes its analoguesand derivatives. For instance, the natural nucleobases, adenine (A) andguanine (G). Other examples of naturally occurring purine derivativesare hypoxanthine, xanthine, theobromine, caffeine, uric acid, andisoguanine. Exemplary purines and pyrimidines include those disclosed inU.S. Pat. No. 3,687,808; Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859; Kroschwitz, J. I., ed. John Wiley & Sons,1990; and Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613, each of which is hereby incorporated by reference in itsentirety.

The term “nucleobase” includes all natural and synthetic nucleobases aswell as universal nucleobases. Typical natural nucleobases includeadenine, guanine, cytosine, uracil, and thymine. Synthetic nucleobasestypically include inosine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine. As used herein, a universal nucleobase isany modified, unmodified, naturally occurring or non-naturally occurringnucleobase that can substitute for more than one of the naturalnucleobases. Universal bases typically contain an aromatic ring moietythat may or may not contain nitrogen atoms and generally use aromaticring stacking to stabilize an oligonucleotide duplex. Some universalbases can be covalently attached to the C-1′ carbon of a pentose sugarto make a universal nucleotide. Some universal bases do not hydrogenbond specifically with another nucleobase. Some universal bases basepair with all of the naturally occurring nucleobases. Some universalbases may interact with adjacent nucleotide bases on the same nucleicacid strand by hydrophobic stacking Exemplary universal nucleobasesinclude, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl,nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazle,4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl,6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl,pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl,propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl,4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl,pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivativesthereof.

Suitable nucleobases include, but are not limited to, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, and O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808; Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859; Kroschwitz, J. I., ed. John Wiley & Sons,1990; and Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613, each of which is hereby incorporated by reference in itsentirety.

The term “nucleoside” refers to a compound comprising a nucleobase, asdefined herein, linked to a pentose at the 1′-position. When thenucleobase is a purine derivative or anologue, the pentose is typicallyattached to the nucleobase at the 9-position of the purine derivative oranologue. When the nucleobase is a pyrimidine derivative or anologue,the pentose is typically attached to the nucleobase at the 1-position ofthe pyrimidine (e.g., Kornberg and Baker, DNA Replication, 2nd Ed.,Freeman, San Francisco, 1992, which is hereby incorporated by referencein its entirety). When a nucleoside is present in R³, R⁴, or R⁵ herein,the nucleoside may be connected to the neighboring atom(s) through anyatom on the nucleobase or pentose.

The term “fatty acid” generally refers to a carboxylic acid with analiphatic tail (chain). The aliphatic chain can be between about 2 andabout 36 carbon atoms in length. Fatty acids can be saturated,unsaturated, or polyunsaturated. The aliphatic chain can be a linear ora branched chain. The term “fatty acid” may be used herein to refer to a“fatty acid derivative” which can include one or more different fattyacid derivatives, or mixtures of fatty acids derivatives. Exemplaryfatty acids include unsaturated fatty acids, saturated fatty acids, anddiacids; mono-, di-, and tri-glycerides of ascarosides that have acarboxylic acid functionality; hydroxy acids, ω hydroxy acids, ω-1hydroxy acids, di-hydroxy fatty acids (e.g., dihydroxy fatty acids thatare omega- or omega-1 hydroxylated, as well as alpha- orbeta-hydroxylated fatty acids).

The term “sugar” refers to a compound which is either a carbohydrate perse made up of one or more monosaccharide units having at least 5 carbonatoms (which may be linear, branched, or cyclic) with an oxygen,nitrogen, or sulfur atom bonded to each carbon atom; or a compoundhaving as a part thereof a carbohydrate moiety made up of one or moremonosaccharide units each having at least 5 carbon atoms (which may belinear, branched or cyclic), with an oxygen, nitrogen or sulfur atombonded to each carbon atom. Representative sugars include the mono-,di-, tri-, and oligosaccharides containing from about 4-9 monosaccharideunits, and polysaccharides such as starches, glycogen, cellulose, andpolysaccharide gums. Exemplary monosaccharides include C₅ and above(e.g., C₅-C₈ or C₅-C₆) sugars; di- and trisaccharides include sugarshaving two or three monosaccharide units (e.g., C₅-C₈ or C₅-C₈).

The term “monosaccharide” means a sugar molecule having a chain of 3-10carbon atoms in the form of an aldehyde (aldose) or ketone (ketose).Suitable monosaccharides include both naturally occurring and syntheticmonosaccharides. Suitable monosaccharides include trioses, such asglycerone and dihydroxyacetone; textroses such as erythrose anderythrulose; pentoses, such as xylose, arabinose, ribose, xyluloseribulose; methyl pentoses (6-deoxyhexoses), such as rhamnose and fucose;hexoses, such as ascarylose, glucose, mannose, galactose, fructose, andsorbose; and heptoses, such as glucoheptose, galamannoheptose,sedoheptulose, and mannoheptulose. Exemplary monosaccharides embraceradicals of allose, altrose, arabinose, cladinose, erythrose,erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose,fuculose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine,galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose,glucose-6-phosphate, gulose glyceraldehyde, L-glycero-D-mannos-heptose,glycerol, glycerone, gulose, idose, lyxose, mannosamine, mannose,mannose-6-phosphate, psicose, quinovose, quinovasamine, rhamnitol,rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose,tagatose, talose, tartaric acid, threose, xylose, and xylulose. Themonosaccharide can be in D- or L-configuration. A typical monosaccharideused herein is hexose.

The monosaccharide may further be a deoxy sugar (alcoholic hydroxy groupreplaced by hydrogen), amino sugar (alcoholic hydroxy group replaced byamino group), a thio sugar (alcoholic hydroxy group replaced by thiol,or C═O replaced by C═S, or a ring oxygen of cyclic form replaced bysulfur), a seleno sugar, a telluro sugar, an aza sugar (ring carbonreplaced by nitrogen), an imino sugar (ring oxygen replaced bynitrogen), a phosphano sugar (ring oxygen replaced with phosphorus), aphospha sugar (ring carbon replaced with phosphorus), a C-substitutedmonosaccharide (hydrogen at a non-terminal carbon atom replaced withcarbon), an unsaturated monosaccharide, an alditol (carbonyl groupreplaced with CHOH group), aldonic acid (aldehydic group replaced bycarboxy group), a ketoaldonic acid, a uronic acid, an aldaric acid, andso forth. Amino sugars include amino monosaccharides, such asgalactosamine, glucosamine, mannosamine, fucosamine, quinovasamine,neuraminic acid, muramic acid, lactosediamine, acosamine, bacillosamine,daunosamine, desosamine, forosamine, garosamine, kanosamine,kansosamine, mycaminose, mycosamine, perosamine, pneumosamine,purpurosamine, rhodosamine. It is understood that the monosaccharide andthe like can be further substituted.

The terms “disaccharide”, “trisaccharide”, and “polysaccharide” embraceradicals of abequose, acrabose, amicetose, amylopectin, amylose, apiose,arcanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose,cellulose, chacotriose, chalcose, chitin, colitose, cyclodextrin,cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, diginose, digitalose,digitoxose, evalose, evemitrose, fructoologosachharide,galto-oligosaccharide, gentianose, gentiobiose, glucan, glucogen,glycogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose,isomaltotriose, isopanose, kojibiose, lactose, lactosamine,lactosediamine, laminarabiose, levoglucosan, levoglucosenone, β-maltose,maltriose, mannan-oligosaccharide, manninotriose, melezitose, melibiose,muramic acid, mycarose, mycinose, neuraminic acid, nigerose,nojirimycin, moviose, oleandrose, panose, paratose, planteose,primeverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose,solatriose, sophorose, stachyose, streptose, sucrose, α,α-trehalose,trehalosamine, turanose, tyvelose, xylobiose, umbelliferose, and thelike. Further, it is understood that the “disaccharide”,“trisaccharide”, and “polysaccharide” and the like can be furthersubstituted. Disaccharide also includes amino sugars and theirderivatives, particularly, a mycaminose derivatized at the C-4′ positionor a 4 deoxy-3-amino-glucose derivatized at the C-6′ position.

The term “polycyclic” or “multi-cyclic” used herein indicates amolecular structure having two or more rings, including, but not limitedto, fused, bridged, or spiro rings.

The above “alkyl”, “alkenyl”, “cycloalkyl”, and “cycloalkenyl” radicals,as well as the ring system of the above aryl, heterocyclyl, orheteroaryl groups, may be optionally substituted.

The term “substituted” or “optionally substituted” is used to indicatethat a group may have a substituent at each substitutable atom of thegroup (including more than one substituent on a single atom), providedthat the designated atom's normal valency is not exceeded and theidentity of each substituent is independent of the others. In accordancewith the present invention, up to three H atoms in each residue can bereplaced with alkyl, halogen, haloalkyl, alkyenyl, haloalkenyl,cycloalkyl, cycloalkenyl, hydroxy, alkoxy, acyl, carboxy, carboalkoxy(also referred to as alkoxycarbonyl), carboxamido (also referred to asalkylaminocarbonyl), cyano, carbonyl, nitro, amino, alkylamino,dialkylamino, mercapto, alkylthio, sulfoxide, sulfone, acylamino,amidino, aryl, heteroaryl, heterocyclyl, aryloxy, heteroaryloxy, apurine or pyridimine or an analogue or derivative thereof (as defined in“nucleobase”), or a sugar such as a monosaccharide having 5 or 6 carbonatoms (as defined in “monosaccharide”). “Unsubstituted” atoms bear allof the hydrogen atoms dictated by their valency. When a substituent isketo (i.e., ═O), then two hydrogens on the atom are replaced.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds; by “stable compound” or“stable structure” is meant a compound that is sufficiently robust tosurvive isolation to a useful degree of purity from a reaction mixture,and formulation into an efficacious agent.

In the characterization of some of the substituents, certainsubstituents may combine to form rings. Unless stated otherwise, it isintended that such rings may exhibit various degrees of unsaturation(from fully saturated to fully unsaturated), may include heteroatoms,and may be substituted with other substituent groups as described above.

The compounds described herein may contain one or more asymmetriccenters and may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms. Each chiral center may be defined, in terms ofabsolute stereochemistry, as (R)- or (S)-. The present invention ismeant to include all such possible isomers, as well as mixtures thereof,including racemic and optically pure forms. Optically active (R)- and(S)-, (−)- and (+)-, or (D)- and (L)-isomers may be prepared usingchiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic doublebonds or other centers of geometric asymmetry, and unless specifiedotherwise, it is intended that the compounds include both E and Zgeometric isomers. Likewise, all tautomeric forms are also intended tobe included. The configuration of any carbon-carbon double bondappearing herein is selected for convenience only and is not intended todesignate a particular configuration; thus a carbon-carbon double bonddepicted arbitrarily herein as trans may be Z, E, or a mixture of thetwo in any proportion.

The term “compounds of the invention,” and equivalent expressions, aremeant to embrace the prodrugs, the pharmaceutically acceptable salts,the oxides, the solvates, e.g. hydrates, and inclusion complexes of thatcompound, where the context so permits, as well as any stereoisomericform, or a mixture of any such forms of that compound in any ratio,unless otherwise specified. Inclusion complexes are described inRemington, The Science and Practice of Pharmacy, 19th Ed. 1:176-177(1995), which is hereby incorporated by reference in its entirety. Themost commonly employed inclusion complexes are those with cyclodextrins,and all cyclodextrin complexes, natural and synthetic, are specificallyencompassed within the claims. Thus, in accordance with some embodimentsof the invention, a compound as described herein, including in thecontexts of pharmaceutical compositions, methods of treatment, andcompounds per se, is provided as the salt form. Similarly, reference tointermediates, whether or not they themselves are claimed, is meant toembrace their salts, and solvates, where the context so permits. For thesake of clarity, particular instances when the context so permits aresometimes indicated in the text, but these instances are purelyillustrative and it is not intended to exclude other instances when thecontext so permits.

The “quaternization” of any basic nitrogen-containing groups of thecompounds disclosed herein is also contemplated. The basic nitrogen canbe quaternized with any agents known to those of ordinary skill in theart including, for example, lower alkyl halides, such as methyl, ethyl,propyl and butyl chloride, bromides and iodides; dialkyl sulfatesincluding dimethyl, diethyl, dibutyl and diamyl sulfates; long chainhalides such as decyl, lauryl, myristyl and stearyl chlorides, bromidesand iodides; and aralkyl halides including benzyl and phenethylbromides. Water or oil-soluble or dispersible products may be obtainedby such quaternization.

As used herein, the term “ascaroside” refers to a compound of Formula I:

or a pharmaceutical equivalent, derivative, analog, and/or salt thereof.As readily apparent to one of skill in the art, the compound may befurther defined by various R groups. For example, in accordance with anembodiment herein, R² may contain the moiety of the formula:

or a pharmaceutical equivalent, derivative, analog and/or salt thereof.

As used herein, the term “ascr” and the designated # refers to varietiesof ascarosides and ascaroside-like compounds. For example, ascr#1 is acompound described herein as a Formula III:

or a pharmaceutical equivalent, derivative, analog and/or salt thereof.

As disclosed herein, the inventors performed a liquid chromatographymass spectrometric screen for ascarosides in 21 different nematodespecies, both free-living and parasitic. It was found that many speciesproduce species-specific but partially overlapping blends ofascarosides, indicating that ascarosides are highly conserved amongnematodes. The inventors found that many nematodes also respond toascarosides through attraction or repulsion, presenting possible newtargets for the control of parasites. Additionally, as disclosed herein,the inventors also investigated the effect of ascarosides on a mouseasthma model. They found that administration of ascaroside compounds,specifically administration of ascr#1, ascr#3 and ascr#7, which wasproduced in Nippostrongylus brasiliensis, resulted in a reduction ofeosinophils, decreased cellular infiltration and decreased mucushyper-production in asthmatic mice.

In one embodiment, the present invention provides a method of treating,alleviating and/or preventing a disorder in a subject by administering acomposition comprising a therapeutically effective dosage of a compoundof Formula I:

or a pharmaceutical equivalent, derivative, analog, and/or salt thereof.In another embodiment, the disorder is an inflammatory and/or immunedisorder. In another embodiment, the disorder is acne vulgaris, asthma,autoimmune diseases, celiac disease, chronic prostatitis,glomerulonephritis, Hypersensitivities, Inflammatory bowel diseases,pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis,sarcoidosis, transplant rejection, vasculitis, Interstitial cystitis,lupus, scleroderma, certain types of hemolytic anemia, type onediabetes, graves disease, multiple sclerosis, Goodpasture's syndrome,pernicious anemia, some types of myopathy, seasonal allergy,mastocytosis, perennial allergy, anaphylaxis, food allergy, allergicrhinitis, atopic dermatitis, and/or autism. In another embodiment, thecomposition is administered orally. In another embodiment, thecomposition is administered by direct injection and/or intravenously. Inanother embodiment, the subject is a human. In another embodiment, thesubject is a rodent. In another embodiment, the subject is selected fromthe group consisting of primates, equines, horses, cattle, cows, swine,sheep, rats, pets, cats, dogs and guinea pigs.

In another embodiment, the present invention provides a compositioncomprising a therapeutically effective dosage of a compound of FormulaI:

or a pharmaceutical equivalent, derivative, analog, and/or salt thereof,and a pharmaceutically acceptable carrier. In another embodiment, thecomposition comprises a blend of ascr#1, ascr#3 and/or ascr#7.

In accordance with various embodiments herein, additional compoundsdesignated as R groups may further define the compound of Formula I:

Various embodiments include where:

-   R¹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl, a haloalkyl, an    alkenyl, or a haloalkenyl; where each R is independently H, halogen,    an alkyl, or an alkenyl;-   R^(1′) is absent, H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl, a    haloalkyl, an alkenyl, or a haloalkenyl; where each R is    independently H, halogen, an alkyl, or an alkenyl;-   R² is a moiety of formula

where:

-   -   each R¹ is independently H, —C(R)₃, —OR, —N(R)₂, halogen, an        alkyl, a haloalkyl, an alkenyl, or a haloalkenyl; where each R        is independently H, halogen, an alkyl, or an alkenyl;    -   R⁵ is H, —OH, —OR⁶, —OCR⁶R⁷R⁸, —CR⁶R⁷R⁸, —NH₂, —NHR⁶, —NR⁶R⁷,        halogen, an alkyl, a haloalkyl, an alkenyl, a haloalkenyl, an        aryl, a heteroaryl, an arylalkyl, a heterocyclyl, a cycloalkyl,        a cycloalkenyl, an acyl, an amino acid, a nucleoside, a        monosaccharide having 5 or 6 carbon atoms, or a bond connecting        to R³ or R⁴ of another unit of Formula I;        -   where:        -   R⁶ and R⁷ are each independently H, —CR₃, —OR, —N(R)₂,            halogen, an alkyl, a haloalkyl, an alkenyl, a haloalkenyl,            an aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, or a            cycloalkenyl, where the alkyl, alkenyl, aryl, heteroaryl,            heterocyclyl, cycloalkyl, or cycloalkenyl is optionally            substituted with one or more substituents independently            selected from the group consisting of —OR⁸, —C(O)R⁸,            —NHC(O)R⁸, an alkyl, a haloalkyl, an aryl, a heteroaryl, a            heterocyclyl, and a cycloalkyl;            -   where:            -   each R is independently H, halogen, an alkyl, or an                alkenyl; and R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹,                —[C(R)₂]_(n4)C(O)(NH)R⁹, —OR, —N(R)₂, halogen, an alkyl,                a haloalkyl, an alkenyl, a haloalkenyl, an aryl, a                heteroaryl, a heterocyclyl, a cycloalkyl, a                cycloalkenyl, a purine, a pyrimidine, or a                monosaccharide having 5 or 6 carbon atoms, where the                alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,                cycloalkyl, cycloalkenyl, purine, pyrimidine, or                monosaccharide is optionally substituted with one or                more substituents independently selected from the group                consisting of —C(R)₃, —OR⁹, —C(O)R⁹, —NHC(O)R⁹, halogen,                an alkyl, a haloalkyl, an aryl, a heteroaryl, a                heterocyclyl, a cycloalkyl, and a monosaccharide having                5 or 6 carbon atoms;                -   where:                -   each R is independently H, halogen, an alkyl, or an                    alkenyl; R⁹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an                    alkyl, a haloalkyl, an alkenyl, a haloalkenyl, an                    aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, a                    cycloalkenyl, a purine, a pyrimidine, or a                    monosaccharide having 5 or 6 carbon atoms, where the                    alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,                    cycloalkyl, cycloalkenyl, purine, pyrimidine, or                    monosaccharide is optionally substituted with one or                    more substituents independently selected from the                    group consisting of —C(R)₃, —OR, —C(O)R, halogen, an                    alkyl, a haloalkyl, an aryl, a heteroaryl, a                    heterocyclyl, and a cycloalkyl; where each R is                    independently H, halogen, an alkyl, or an alkenyl;                    and                -   n⁴ n is an integer of 1 to 30; and        -   R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹,            —[C(R)₂]_(n4)C(O)(NH)R⁹, —OR, —N(R)₂, halogen, an alkyl, a            haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl,            a heterocyclyl, a cycloalkyl, a cycloalkenyl, a purine, a            pyrimidine, or a monosaccharide having 5 or 6 carbon atoms,            where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,            cycloalkyl, cycloalkenyl, purine, pyrimidine, or            monosaccharide is optionally substituted with one or more            substituents independently selected from the group            consisting of —C(R)₃, —OR⁹, —C(O)R⁹, —NHC(O)R⁹, halogen, an            alkyl, a haloalkyl, an aryl, a heteroaryl, a heterocyclyl, a            cycloalkyl, and a monosaccharide having 5 or 6 carbon atoms;            -   where:            -   each R is independently H, halogen, an alkyl, or an                alkenyl;            -   R⁹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl, a                haloalkyl, an alkenyl, a haloalkenyl, an aryl, a                heteroaryl, a heterocyclyl, a cycloalkyl, a                cycloalkenyl, a purine, a pyrimidine, or a                monosaccharide having 5 or 6 carbon atoms, where the                alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,                cycloalkyl, cycloalkenyl, purine, pyrimidine, or                monosaccharide is optionally substituted with one or                more substituents independently selected from the group                consisting of —C(R)₃, —OR, —C(O)R, halogen, an alkyl, a                haloalkyl, an aryl, a heteroaryl, a heterocyclyl, and a                cycloalkyl; where each R is independently H, halogen, an                alkyl, or an alkenyl; and            -   n⁴ is an integer of 1 to 30;    -   n¹, n², and n³ are each independently an integer of 0 to 30;    -   n⁴ is an integer of 1 to 30; and    -   the sum of n¹, each n², and each n³ is 1 to 30;

-   R³ and R⁴ are each independently H, —CR⁶R⁷R⁸, —C(O)R⁸, an alkyl, a    haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl, a    heterocyclyl, a cycloalkyl, a cycloalkenyl, an acyl, an amino acid,    a nucleoside, a monosaccharide having 5 or 6 carbon atoms, or a bond    connecting to R⁵ of another unit of Formula I; where:    -   R⁶ and R⁷ are each independently H, —CR₃, —OR, —N(R)₂, halogen,        an alkyl, a haloalkyl, an alkenyl, a haloalkenyl, an aryl, a        heteroaryl, a heterocyclyl, a cycloalkyl, or a cycloalkenyl,        where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,        cycloalkyl, or cycloalkenyl is optionally substituted with one        or more substituents independently selected from the group        consisting of —OR⁸, —C(O)R⁸, —NHC(O)R⁸, an alkyl, a haloalkyl,        an aryl, a heteroaryl, a heterocyclyl, and a cycloalkyl;        -   where:        -   each R is independently H, halogen, an alkyl, or an alkenyl;            and        -   R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹,            —[C(R)₂]_(n4)C(O)(NH)R⁹, —OR, —N(R)₂, halogen, an alkyl, a            haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl,            a heterocyclyl, a cycloalkyl, a cycloalkenyl, a purine, a            pyrimidine, or a monosaccharide having 5 or 6 carbon atoms,            where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,            cycloalkyl, cycloalkenyl, purine, pyrimidine, or            monosaccharide is optionally substituted with one or more            substituents independently selected from the group            consisting of —C(R)₃, —OR⁹, —C(O)R⁹, —NHC(O)R⁹, halogen, an            alkyl, a haloalkyl, an aryl, a heteroaryl, a heterocyclyl, a            cycloalkyl, and a monosaccharide having 5 or 6 carbon atoms;            -   where:            -   each R is independently H, halogen, an alkyl, or an                alkenyl; R⁹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an                alkyl, a haloalkyl, an alkenyl, a haloalkenyl, an aryl,                a heteroaryl, a heterocyclyl, a cycloalkyl, a                cycloalkenyl, a purine, a pyrimidine, or a                monosaccharide having 5 or 6 carbon atoms, where the                alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,                cycloalkyl, cycloalkenyl, purine, pyrimidine, or                monosaccharide is optionally substituted with one or                more substituents independently selected from the group                consisting of —C(R)₃, —OR, —C(O)R, halogen, an alkyl, a                haloalkyl, an aryl, a heteroaryl, a heterocyclyl, and a                cycloalkyl; where each R is independently H, halogen, an                alkyl, or an alkenyl; and            -   n⁴ is an integer of 1 to 30; and    -   R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹, —[C(R)₂]_(n4)C(O)(NH)R⁹,        —OR, —N(R)₂, halogen, an alkyl, a haloalkyl, an alkenyl, a        haloalkenyl, an aryl, a heteroaryl, a heterocyclyl, a        cycloalkyl, a cycloalkenyl, a purine, a pyrimidine, or a        monosaccharide having 5 or 6 carbon atoms, where the alkyl,        alkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,        cycloalkenyl, purine, pyrimidine, or monosaccharide is        optionally substituted with one or more substituents        independently selected from the group consisting of —C(R)₃,        —OR⁹, —C(O)R⁹, —NHC(O)R⁹, halogen, an alkyl, a haloalkyl, an        aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, and a        monosaccharide having 5 or 6 carbon atoms;        -   where:        -   each R is independently H, halogen, an alkyl, or an alkenyl;        -   R⁹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl, a            haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl,            a heterocyclyl, a cycloalkyl, a cycloalkenyl, a purine, a            pyrimidine, or a monosaccharide having 5 or 6 carbon atoms,            where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,            cycloalkyl, cycloalkenyl, purine, pyrimidine, or            monosaccharide is optionally substituted with one or more            substituents independently selected from the group            consisting of —C(R)₃, —OR, —C(O)R, halogen, an alkyl, a            haloalkyl, an aryl, a heteroaryl, a heterocyclyl, and a            cycloalkyl; where each R is independently H, halogen, an            alkyl, or an alkenyl; and        -   n⁴ is an integer of 1 to 30; and

-   each R⁵ is independently H, —OH, —OR⁶, —OCR⁶R⁷R⁸, —CR⁶R⁷R⁸, —NH₂,    —NHR⁶, —NR⁶R⁷, halogen, an alkyl, a haloalkyl, an alkenyl, a    haloalkenyl, an aryl, a heteroaryl, an arylalkyl, a heterocyclyl, a    cycloalkyl, a cycloalkenyl, an acyl, an amino acid, a nucleoside, a    monosaccharide having 5 or 6 carbon atoms, or a bond connecting to    R³ or R⁴ of another unit of Formula I;    -   where:    -   R⁶ and R⁷ are each independently H, —CR₃, —OR, —N(R)₂, halogen,        an alkyl, a haloalkyl, an alkenyl, a haloalkenyl, an aryl, a        heteroaryl, a heterocyclyl, a cycloalkyl, or a cycloalkenyl,        where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,        cycloalkyl, or cycloalkenyl is optionally substituted with one        or more substituents independently selected from the group        consisting of —OR⁸, —C(O)R⁸, —NHC(O)R⁸, an alkyl, a haloalkyl,        an aryl, a heteroaryl, a heterocyclyl, and a cycloalkyl;        -   where:        -   each R is independently H, halogen, an alkyl, or an alkenyl;            and        -   R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹,            —[C(R)₂]_(n4)C(O)(NH)R⁹, —OR, —N(R)₂, halogen, an alkyl, a            haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl,            a heterocyclyl, a cycloalkyl, a cycloalkenyl, a purine, a            pyrimidine, or a monosaccharide having 5 or 6 carbon atoms,            where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,            cycloalkyl, cycloalkenyl, purine, pyrimidine, or            monosaccharide is optionally substituted with one or more            substituents independently selected from the group            consisting of —C(R)₃, —OR⁹, —C(O)R⁹, —NHC(O)R⁹, halogen, an            alkyl, a haloalkyl, an aryl, a heteroaryl, a heterocyclyl, a            cycloalkyl, and a monosaccharide having 5 or 6 carbon atoms;            -   where:            -   each R is independently H, halogen, an alkyl, or an                alkenyl;            -   R⁹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl, a                haloalkyl, an alkenyl, a haloalkenyl, an aryl, a                heteroaryl, a heterocyclyl, a cycloalkyl, a                cycloalkenyl, a purine, a pyrimidine, or a                monosaccharide having 5 or 6 carbon atoms, where the                alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,                cycloalkyl, cycloalkenyl, purine, pyrimidine, or                monosaccharide is optionally substituted with one or                more substituents independently selected from the group                consisting of —C(R)₃, —OR, —C(O)R, halogen, an alkyl, a                haloalkyl, an aryl, a heteroaryl, a heterocyclyl, and a                cycloalkyl; where each R is independently H, halogen, an                alkyl, or an alkenyl; and            -   n⁴ is an integer of 1 to 30; and    -   R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹, —[C(R)₂]_(n4)C(O)(NH)R⁹,        —OR, —N(R)₂, halogen, an alkyl, a haloalkyl, an alkenyl, a        haloalkenyl, an aryl, a heteroaryl, a heterocyclyl, a        cycloalkyl, a cycloalkenyl, a purine, a pyrimidine, or a        monosaccharide having 5 or 6 carbon atoms, where the alkyl,        alkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,        cycloalkenyl, purine, pyrimidine, or monosaccharide is        optionally substituted with one or more substituents        independently selected from the group consisting of —C(R)₃,        —OR⁹, —C(O)R⁹, —NHC(O)R⁹, halogen, an alkyl, a haloalkyl, an        aryl, a heteroaryl, a heterocyclyl, a cycloalkyl, and a        monosaccharide having 5 or 6 carbon atoms;        -   where:        -   each R is independently H, halogen, an alkyl, or an alkenyl;        -   R⁹ is H, —C(R)₃, —OR, —N(R)₂, halogen, an alkyl, a            haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl,            a heterocyclyl, a cycloalkyl, a cycloalkenyl, a purine, a            pyrimidine, or a monosaccharide having 5 or 6 carbon atoms,            where the alkyl, alkenyl, aryl, heteroaryl, heterocyclyl,            cycloalkyl, cycloalkenyl, purine, pyrimidine, or            monosaccharide is optionally substituted with one or more            substituents independently selected from the group            consisting of —C(R)₃, —OR, —C(O)R, halogen, an alkyl, a            haloalkyl, an aryl, a heteroaryl, a heterocyclyl, and a            cycloalkyl; where each R is independently H, halogen, an            alkyl, or an alkenyl; and        -   n⁴ is an integer of 1 to 30; or            (ii) a compound comprising:    -   at least one nucleobase,    -   at least one fatty acid,    -   at least one amino acid, and    -   at least one sugar;    -   where the at least one nucleobase, the at least one fatty acid,        the at least one amino acid, and the at least one sugar are        linked by covalent bonds; and    -   where the compound has a molecular weight of less than about        2,000 g/mol.

In at least one embodiment of this aspect of the present invention, theone or more modulator compounds is a compound of Formula I. Suitablemodulator compounds according to this embodiment include one or moreisolated modulator compounds of Formula I′ or Formula I″:

Suitable modulator compounds according to this embodiment also includeone or more isolated modulator compounds of Formula I in which at leastone of the following conditions is met:

-   (i) R¹ is independently —C(R′)₃, —OR, —N(R)₂, halogen, a haloalkyl,    an alkenyl, or a haloalkenyl; wherein each R is independently H,    halogen, an alkyl, or an alkenyl, and each R′ is independently    halogen or an alkenyl; and    -   R^(1′) is independently absent, H, —C(R)₃, —OR, —N(R)₂, halogen,        an alkyl, a haloalkyl, an alkenyl, or a haloalkenyl;-   (ii) R² is

-   -   wherein:    -   each        is independently a single or double bond;    -   q¹, q², and q³ are each independently an integer of 1 to 26;    -   q⁴ and q⁵ are each independently an integer of 0 to 26;    -   the sum of q¹, q², q³, q⁴, and q⁵ is less than or equal to 28;        and    -   the sum of q⁴ and q⁵ is greater than or equal to 2;

-   (iii) R⁵ is H, —OR⁶, —OCR⁶R⁷R⁸, —CR⁶R⁷R⁸, —NH₂, —NR⁶R⁷, halogen, a    haloalkyl, an alkenyl, a haloalkenyl, an aryl, a heteroaryl, an    arylalkyl, a heterocyclyl, a cycloalkyl, a cycloalkenyl, an acyl, an    amino acid, or a nucleoside;

-   (iv) R⁵ is a bond connecting to R³ or R⁴ of another unit of Formula    I, forming a compound containing at least two units of Formula I;

-   (v) R³ and R⁴ are each independently —CR⁶R⁷R⁸, a haloalkyl, an    alkenyl, a haloalkenyl, an aryl, a heteroaryl, a heterocyclyl, a    cycloalkyl, a cycloalkenyl, an acyl, an amino acid, a nucleoside, or    —(CO)R⁸ wherein R⁸ is H, —C(R)₃, —[C(R)₂]_(n4)NHC(O)R⁹,    —[C(R)₂]_(n4)C(O)(NH)R⁹, —OR, —N(R)₂, halogen, an alkyl, a    haloalkyl, a cycloalkyl, a cycloalkenyl, a purine, a pyrimidine, or    a monosaccharide having 5 or 6 carbon atoms, wherein the alkyl,    cycloalkyl, cycloalkenyl, purine, pyrimidine, or monosaccharide is    optionally substituted with one or more substituents independently    selected from the group consisting of —C(R)₃, —OR⁹, —C(O)R⁹,    —NHC(O)R⁹, halogen, an alkyl, a haloalkyl, an aryl, a heteroaryl, a    heterocyclyl, a cycloalkyl, and a monosaccharide having 5 or 6    carbon atoms;

-   (vi) the compound is a compound of Formula I″;    with the proviso that the compound is not

In at least one embodiment of this aspect of the present invention, theone or more modulator compounds is a compound comprising at least onenucleobase, at least one fatty acid, at least one amino acid, and atleast one sugar.

In this and all aspects of the present invention, a single modulatorcompound or a combination of modulator compounds can be administered.

In various aspects of the present invention, ascarosides may be producedby a nematode. The nematode may include Acuarioidea, Aelurostrongylus,Aelurostrongylus abstrusus, Amidostomatidae, Amidostomum, Ancylostomabraziliense, Ancylostoma caninum (dog hookworm), Ancylostoma ceylanicum,Ancylostoma duodenale, Ancylostoma tubaeforme, Ancylostomatidae,Ancylostomatinae, Angiostrongylidae, Angiostrongylus, Aproctoidea,Ascaridia galli, Ascaris lumbricoidies, Ascaris suum, Brevistriatinae,Brugia malayi, Brugia timori, Bunostominae, Camallanoidea, Carolinensisminutus, Chabertia, Chabertia ovina, Chabertiidae, Cloacina,Cloacinidae, Cooperia, Cooperia pectinata, Cooperia punctata,Cooperiidae, Cosmocercoidea, Crenosoma, Crenosomatidae, Cyathostoma,Cyathostominae, Cyclodontostomum, Cylicocyclus nassatus, Cystocaulus,Cystocaulus ocreatus, Deletrocephalidae, Deletrocephalus,Diaphanocephalidae, Diaphanocephaloidea, Dictyocaulidae, Dictyocaulinae,Dictyocaulus arnfeldi, Dictyocaulus filaria, Dictyocaulus osleri,Dictyocaulus viviparus, Dictyocausus viviparous, Didelphostrongylus,Dioctophyma renale, Dioctophymatoidea, Diplotriaenoidea, Dirofilariaimmitis, Dracunculoidea, Dromaeostrongylidae, Elaeophora scheideri,Elaphostrongylinae, Filarinema, Filarioidea, Filaroides, Filaroididae,Gapeworm, Ghathostomatoidea, Globocephaloidinae, Gongylonema pulchrum,Gyalocephalinae, Habronema, Habronematoidea, Haemonchidae, Haemonchinae,Haemonchus contortus, Haemonchus placei, Halocercus, Heligmonellidae,Heligmonellinae, Heligmonoides speciosus, Heligmosomatidae,Heligmosomidae, Heligmosomoidea, Heligmosomoides, Herpetostrongylidae,Herpetostrongylinae, Heterakoidea, Hovorkonema, Hypodontus,Kalicephalus, Labiomultiplex, Labiosimplex, Labiostrongylus,Libyostrongylinae, Loa loa, Longistriata, Mackerrastrongylidae,Macroponema, Macropostrongylus, Mansonella ozzardi, Mansonella perstans,Mansonella streptocerca, Marshallagia, Metastrongylidae,Metastrongyloidea (lungworms), Metastrongyloidea sp. RJ-2010,Metastrongylus, Metastrongylus apri, Metastrongylus asymmetricus,Metastrongylus confusus, Metastrongylus elongates, Metastrongyluspudendotectus, Metastrongylus salmi, Molineidae, Molineoidea,Monilonema, Muellerinae, Muellerius capillaris, Muspiceoidea,Nematodirinae, Nematodirus battus, Neoheligmonella granjoni, Nicollina,Nicollinidae, Nippostrongylinae, Nippostrongylus brasiliensis,Oesophagostomum, Oesophagostomum columbianum, Oesophagostomum radiatum,Ohbayashinema, Oncocerca volvulus, Orientostrongylus ezoensis, Oslerus,Oslerus osleri, Ostertagia ostertagi, Ostertagia venulosum,Otostrongylus, Oxyurodiea, Papillostrongylus, Paraelaphostronyglustenuis, Parafilaroides, Parazoniolaimus, Physalopteroidea,Potorostrongylus, Protostrongylidae, Protostrongylinae, Pseudaliidae,Pseudalius, Rictularioidea, Rugopharynx, Setaria cervi, Seuratoidea,Skrjabingylus, Spiruroidea, Stenurus, Stephanofilaria stilesi,Stephanuridae, Stephanurus, Strongylida, Strongylida sp. AM-2008,Strongylidae, Strongylinae, Strongyloidea, Strongyloides papillosus,Subuluroidea, Syngamidae, Syngamus, Teladorsagia circumcincta,Ternidens, Tetrabothriostrongylus, Thelazioidea, Torynurus, Toxocanacanis, Toxocara cati, Toxocara vitulorum, Trichinella spiralis,Trichinelloidea, Trichostronglyus axei, Trichostronglyus colubriformis,Trichostronglyus vitrinus, Trichostrongylidae, Trichostrongylinae,Trichostrongyloidea, Trichuris suis, Troglostrongylus,Umingmakstrongylus pallikuukensis, Unclassified Metastrongyloidea,unclassified Protostrongylidae, unclassified Strongylida, unclassifiedTrichostrongylidae, Varestrongylinae, Wucheria bancrofti, andZoniolaimus. Other suitable nematodes according to this aspect of thepresent invention include Amidostomatidae, Ancylostomatidae,Angiostrongylidae, Brevistriatinae, Chabertiidae, Cloacinidae,Cooperiidae, Crenosomatidae, Deletrocephalidae, Diaphanocephalidae,Dictyocaulidae, Filaroididae, Haemonchidae, Heligmonellidae,Heligmosomatidae, Heligmosomidae, Herpetostrongylidae,Mackerrastrongylidae, Metastrongylidae, Molineidae, Nicollinidae,Oxyurodidea, Parafilaroidea, Protostrongylidae, Pseudaliidae,Stephanuridae, Strongylidae, Syngamidae, Trichostrongylidae,Ancylostomatinae, Brevistriatinae, Bunostominae, Cyathostominae,Dictyocaulinae, Gyalocephalinae, Haemonchinae, Heligmonellinae,Herpetostrongylinae, Libyostrongylinae, Muellerinae, Nematodirinae,Nippostrongylinae, Protostrongylinae, Strongylinae, Trichostrongylinae,Varestrongylinae, Acuarioidea, Aproctoidea, Camallanoidea,Cosmocercoidea, Diaphanocephaloidea, Dioctophymatoidea,Diplotriaenoidea, Dracunculoidea, Filarioidea, Ghathostomatoidea,Habronematoidea, Heligmosomoidea, Heterakoidea, Metastrongyloidea,Molineoidea, Muspiceoidea, Physalopteroidea, Rictularioidea,Seuratoidea, Spiruroidea, Strongyloidea, Subuluroidea, Thelazioidea,Trichinelloidea, Trichostrongyloidea, Metastrongyloidea,Protostrongylidae, Strongylidae, Trichostrongylidae,Aelurostrongylusabstrusus, Amidostomumanseris, Ancylostomabraziliense,Ancylostomacaninum, Ancylostomaceylanicum, Ancylostomaduodenale,Ancylostomatubaeforme, Angiostrongyluscantonensis, Ascaridiagalli,Ascarislumbricoidies, Ascarissuum, Bolbosomacapitatum, Brugiamalayi,Brugiatimori, Bunostomumphlembotomum, Capillariabovis,Carolinensisminutus, Chabertiaovina, Cloacina sp., Cooperiapectinata,Cooperiapunctata, Cooperia spp., Crassicaudaboopis, Crenosoma sp.,Cyathostoma sp., Cyathostomumcatinatum, Cyathostomumcoronatum,Cyclodontostomum, Cylicocyclusnassatus, Cylicostephanuscalicatus,Cylicostephanusgoldi, Cylicostephanuslongibursatus, Cystocaulus sp.,Cystocaulusocreatus, Deletrocephalus sp., Dictyocaulusarnfeldi,Dictyocaulusfilaria, Dictyocaulusosleri, Dictyocaulusviviparus,Dictyocausus viviparous, Didelphostrongylus, Dioctophymarenale,Dirofilariaimmitis, Dracucunculusmedinensis, Dromaeostrongylidae,Elaeophorascheideri, Elaphostrongylinae, Enterobiusvermicularis,Filarinema sp., Filaroides sp., Globocephaloidinae, Globoderapallida,Gnathostomadoloresi, Gongylonemapulchrum, Habronema sp.,Haemonchuscontortus, Haemonchusplacei, Halocercus sp.,Heligmonoidesspeciosus, Heligmosomoides sp., Heligmosomoidespolygyrus,Hovorkonema sp., Hypodontus sp., Kalicephalus sp., Labiomultiplex sp.,Labiosimplex sp., Labiostrongylus sp., Loa boa, Longistriata sp.,Macroponema sp., Macropostrongylus, Mansonella ozzardi, Mansonellaperstans, Mansonella streptocerca, Marshallagiasp., Metastrongyloideasp. RJ-2010, Metastrongylus sp., Metastrongylusapri, Metastrongylusasymmetricus, Metastrongylus confusus, Metastrongylus elongates,Metastrongybu spudendotectus, Metastrongybussabmi, Monilonema sp.,Muebberiuscapibbaris, Necator americanus, Nematodirusbattus, Nematodirushelvetianus, Neoheligmonella granjoni, Nicollina sp., Nippostrongybusbrasiliensis, Oesophagostomum sp., Oesophagostomumcolumbianum,Oesophagostomumradiatum, Oesophatostomumdentatum, Ohbayashinema sp.,Onchocerca volvulus, Orientostrongylusezoensis, Oslerus sp.,Oslerusosleri, Ostertagiabisonis, Ostertagiaostertagi,Ostertagiaostertagi, Ostertagiavenulosum, Otostrongylus,Papillostrongylus sp., Paraelaphostronyglus tenuis, Parazoniolaimus sp.,Placentonema gigantissima, Potorostrongylus sp., Pseudalius sp.,Rugopharynx sp., Setariacervi, Skrjabingylus sp., Stenurus sp.,Stephanofilariastilesi, Stephanurus sp., Strongylida sp. AM-2008,Strongyloides papillosus, Strongyloides ratti, Strongyloidesvenezuelensis, Syngamus trachea, Teladorsagia sp., Teladorsagiacircumcincta, Ternidens sp., Tetrabothriostrongylus sp., Torynurus sp.,Toxocanacanis, Toxocaracati, Toxocaravitulorum, Trichinellabritovi,Trichinellamurrelli, Trichinellanativa, Trichinella nelsoni, Trichinellapapuae, Trichinella pseudospiralis, Trichinella spiralis, Trichinellazimbabwensis, Trichostronglyus axei, Trichostronglyus colubriformis,Trichostronglyus vitrinus, Trichostrongylus axei, Trichuris discolor,Trichuris muris, Trichuris suis, Trichuris trichiura, Troglostrongylus,Umingmakstrongylus pallikuukensis, Uncinariastenocephala, Wucheriabancrofti, and Zoniolaimus sp.

EXAMPLES

The following example is provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Generally

Nematodes cause significant human disease and agricultural damage.Interfering with chemically-mediated nematode behaviors couldpotentially prevent or mitigate nematode infections. Two chemicallydistinct mate-finding cues have been reported in nematodes: vanillicacid in Heterodera glycines and several ascarosides in Caenorhabditiselegans. To obtain a general picture of nematode sex pheromones, wepurified the female sex pheromone of the sour-paste nematode,Panagrellus redivivus, and found that it is the ascaroside, ascr#1. Thesame chemical in C. elegans weakly induces diapause, suggesting thatascarosides might be a general class of nematode pheromones. Thus, theinventors performed a liquid chromatography mass spectrometric screenfor ascarosides in 21 different nematode species, both free-living andparasitic. Many species produce species-specific but partiallyoverlapping blends of ascarosides, indicating that ascarosides arehighly conserved among nematodes. The inventors found that manynematodes also respond to ascarosides through attraction or repulsion,presenting possible new targets for the control of parasites.

Nematodes are the most abundant animals in the world. They have beenfound to inhabit sulfurous sediment, deep-sea trenches, human lymphnodes, pig intestines, plant roots, whale placenta, arctic ice, and manyother ecosystems, making them one of the most successful groups ofanimals on earth. Nematodes are responsible for significant crop losses(˜$100 billion annually) and most neglected tropical diseases (severalhundred million affected individuals). Therefore it is no surprise thatmany nematode matefinding behaviors have been studied, in part towardsthe goal of developing methods for parasitic control. These studiescollectively reveal that males from many nematode species are able totrack females in environments as diverse as in plant roots, bacterialfilms, sand, agar, and mammalian intestines. There have also been manyattempts to identify nematode pheromones, but identification has onlybeen successful in two nematode species: the economically devastatingsoybean pathogen Heterodera glycines 26 and Caenorhabditis elegans.Since this discovery, chemical analogs of the H. glycines sex pheromone,vanillic acid has demonstrated potential to reduce parasitic load inaffected crops by preventing males from finding their mates. Studies inthe soil dwelling model organism C. elegans have shown that a family ofsmall-molecule pheromones, called ascarosides, regulates gender specificattraction, repulsion, aggregation, olfactory plasticity, and entry intodauer (a stress-resistant life stage). These ascaroside studies wereamong the first to integrate modern advancements in analytical chemistrywith the wealth of genetic, cellular, and developmental data availablefor C. elegans.

As further described herein, the inventors sought to purify pheromonesfrom a diverse range of nematode species. Starting with the double-blindpurification of the female sex pheromone in the sour-paste nematodePanagrellus redivivus, the inventors found that only one compound servedas the male-attractant in the study, which the inventors then identifiedas ascr#1. Because ascr#1 is known for playing a small role in dauerinduction in C. elegans, it is notable that the inventors found the samecompound played a role in mate-finding in P. redivivus. Thus, theinventors believed that ascarosides are produced ubiquitously bynematodes.

For further investigation, the inventors collected wormexcretions/secretions (E/S) from a wide range of both free-living andparasitic (plant, insect, and mammal) nematode species and screened forascarosides in nematode byproducts. The inventors found that manynematodes indeed produce a wide range of ascarosides and that nematodesfrom several different genera are attracted to many of the sameascarosides, revealing that ascarosides are used by nematodes tocommunicate among species despite the fact that they occupy strikinglydifferent ecologies. This universality presents a new paradigm inunderstanding of nematode communication, as this provides evidence ofcross-species communication among members of this highly diversemetazoan phylum.

Example 2 The Panagrellus redivivus Female Sex Pheromone is anAscaroside

The inventors developed several criteria for the nematode species thatwould ultimately be chosen for investigation for the activity-guidedpheromone purification. The inventors wanted a nematode species thatbelonged to a different Glade than the species that have already hadtheir pheromones identified (H. glycines and C. elegans). Given thatactivity-guided fractionation schemes have many involved steps andrequire many comparisons of nematode response to subtly differentfractions, the inventors also wanted a nematode species that performswell on bioassays. For many of the species that the inventors hadinitially screened (Koernia sp., Pristionchus pacificus, Pristionchuspauli, Acrobeles sp., and Zeldia sp) individuals would move slowly, notat all, or in an inconsistent manner. The inventors chose the sour-pastenematode Panagrellus redivivus because both males and females move welland also because the preliminary experiments showed that both gendersproduce a sex pheromone that attracts the opposite gender. To scorepositive activity in this fractionation scheme, the inventors developeda semi-automated attraction assay to look for attraction to a given cue(FIG. 1). The inventors combined this scoring method with a previouslydescribed fractionation scheme (FIG. 2a ). Specifically,nematode-conditioned media was fractionated based on hydrophobicity(FIG. 2b ) and charge (FIG. 2c ); then two positive fractions werefurther separated by HPLC (FIG. 2d ). Through this process, theinventors discovered a single male-attracting component that was beingproduced by females, but not males. They used nuclear magnetic resonance(NMR) spectroscopy and liquid chromatography mass spectrometry (LCMS) toidentify this compound as (−)6-(3,5-dihydroxy-6-methyltetrahydropyran-2-yloxy) heptanoic acid (akaascr#1, C736 or daumone28) (FIG. 2a ).

To confirm activity of the identified ascaroside, the inventors testedsynthetic samples of ascr#1 to confirm activity and found that P.redivivus males were strongly attracted to synthetic ascr#1 over a widerange of concentrations, with no apparent attenuation of highconcentrations (FIG. 2e ). In this regard, the response of P. redivivusmales to ascr#1 differs from that of C. elegans males to two differentC. elegans hermaphroditic sex pheromones, ascr#2 and ascr#3, as well asunfractionationated hermaphroditie exudates, both of which show abiphasic response with reduced attraction at high concentrations ofascarosides.

Next the inventors tested to see if ascr#1 is a gender-specificpheromone and found that female P. redivivus were not attracted toascr#1 at any of the tested concentrations (FIG. 2e ), allowing theconclusion that ascr#1 is indeed the male-specific attractant. P.redivivus females were not significantly repelled by ascr#1, as measuredby the semi-automated scoring method; however, detailed inspection ofthe videos recorded from the assays revealed that females showed somerepulsive behavior: they tended to stall upon entering the regionholding the ascr#1 sample spot, followed by a change in direction andabrupt exit of the region. Because there is a delay before females exitfrom the ascr#1 sample spot, time spent in the ascr#1 sample spot wascomparable to the time spent in the control region, preventing detectionof repulsion by our automated assay. The inventors thus used a separatemethod for scoring repulsion that focused on behavioral response ratherthan time spent in each region. Defining repulsion as the immediate exitof the conditioned region before entering a full worm's length, theinventors found that females were significantly repelled by highconcentrations of ascr#1. It can be concluded that the P. redivivusfemale sex pheromone is ascr#1, which strongly attracts males but repelsfemales at high concentrations.

Therefore, P. redivivus and C. elegans use structurally related smallmolecules as chemical signals. This result was unexpected given that P.redivivus and C. elegans belong to two different clades of nematodegenera. Because ascr#1 has already been characterized in C. elegans forits limited role in the induction of a diapausal life stage, thefindings demonstrate that the same ascaroside can serve differentfunctions between different species and between different genders.

Example 3 Ascarosides are Broadly Present in Many Nematodes

Based on the conclusion that the C. elegans pheromone, ascr#1, alsoserves as a pheromone in P. redivivus, the inventors believed thatascarosides may represent a general class of nematode signalingmolecules and initiated a mass-spectrometry-based screen for ascarosidesin a larger selection of nematode species. The inventors includednematodes with different types of life histories, including bothparasites (plant, insect and mammal) and free-living nematodes. Thescreen further included species from ancestral Clade 2 and the morerecently evolved Clade 12.

Sample selection was opportunistic in that the inventors used metabolitesamples obtained from mixed populations, such that there could begathered a wide sampling of ascarosides across all larval stages. Formany parasitic nematodes, infective juveniles were separated from adultsto look for differences between chemical cues produced during thehost-seeking life stage and mate-seeking life stage. For metabolitecollection, nematodes were generally incubated with appropriate bufferfor a few hours and then filtered out. The resulting metabolite extractswere analyzed via HPLC-ESI-MS using a protocol specifically optimizedfor the detection of ascarosides in complex metabolome samples. Theseanalyses revealed the presence of ascarosides in most of the nematodesamples. Comparison with mass spectroscopic data and retention times ofa library of 150 ascarosides known from C. elegans led to theidentification of a total of 19 different ascarosides in the 21 speciesanalyzed.

Ascaroside profiles generally varied between species; however, in somecases even entirely unrelated species produced surprisingly similarascaroside mixtures. For example, a highly diverse range of nematodesproduced the saturated ascaroside ascr#10, including the rat parasiticnematode Nippostrongylus brasiliensis, the entomopathogenic insectnematodes Heterorhabditis bacteriophora, Steinernema carpocapsae,Steinernema glaseri, and Steinernema riobrave, the soil nematodeCaenorhabditis elegans, and the necromenic insect parasites Pristionchuspacificus and Koernia sp.

It should be noted that although all of the identified ascarosides havepreviously been found in C. elegans metabolite extracts, most of theseare not produced by C. elegans wild type and were identified frommetabolite extracts of specific C. elegans mutants. This observationsuggests that whereas nematodes have the capability to produce a greatdiversity of ascaroside derivatives, the abundance and proportions ofthe various ascaroside blends are species specific.

Several other species including H. bacteriophora and P. strongyloidespredominantly contained longer chained ascarosides with 11-15 numberedcarbon side-chains. In some cases, ascarosides with saturated 5-, 7-,9-, and 11-numbered carbon side chains, e.g. Caenorhabditis elegans andRhabditis sp. extracts, were accompanied by additional laterelutingisomeric derivatives whose mass spectra suggests that they representderivatives carrying the ascarylose unit on the ultimate carbon of theside chain, instead of the usual penultimate position. The structures ofthese omega-oxygenated ascarosides (“oscr”) was confirmed by synthesisof two representative components featuring five- and nine-carbon sidechains (compounds oscr#9 and oscr#10).

Some of the known ascarosides were not found in some nematode species,namely Pratylenchus species, Heterodera. schactii, Ascaris suum, andRomanomermis species; however, it is possible that these species produceascarosides in amounts smaller than detection limit, or that theirbiosynthesis occurs only under environmental conditions different fromthose in the current study. For example, the parasitic species(Pratylenchus sp. and Heterodera schactii) have a plant-adjacentlifestyle and the Romanomermis species have a mosquito-infective andmoss-dwelling lifestyle that might require collection conditions thatmimic their environments more closely. It is also possible that some ofthe species analyzed produce ascarosides with structural modificationsthat prevented their identification in our mass spectrometric screen.

Finally, the inventors applied the MS screen to Panagrellus redivivus,given that the results for ascr#1 described herein were found usingtraditional activity-guided fractionation. Large quantities of twoascarosides, ascr#1 and ascr#10 in P. redivivus females were found,which were absent in metabolite samples obtained from males. Thisfinding supports the discovery that ascr#1 is indeed a P. redivivusfemale sex pheromone. Following the additional identification of ascr#10in female P. redivivus samples, the inventors tested both males andfemales for attraction by ascr#10. Females do not respond to ascr#10 atany of the concentrations tested; whereas males show some attraction atthe very high, nonphysiological concentration of 1 mM. Because such highconcentrations were not tested during the activity-guided isolation ofthe female pheromone, ascr#10 was not discovered along with ascr#1.

Example 4 Different Species of Nematodes are Attracted to Ascarosides

To investigate the biological function of the ascarosides identified inthe MS screen, the inventors used a bioassay (FIG. 1) to measureattraction of different nematode species to a wide range of ascarosideconcentrations (1 nM, 1 μM, and 1 mM). For these bioassays, nematodespecies had to fulfill several requirements, such as sufficient movementon bacterial lawns, low tendency to aggregate, easy to grow, andgenerally reproducible locomotive behavior. This study was limited to asurvey of males from different species, because males tended to movemuch more evenly across our bioassay lawn than females andhermaphrodites (with the exception of P. redivivus females, which movewell and were therefore in this study.)

The inventors found that males from many of the nematode species testedwere attracted to the same ascarosides, particularly ascr#1, ascr#3,ascr#7, ascr#8, ascr#9, and ascr#10 (FIG. 4). Several nematodes respondto different concentrations of ascarosides, for example P. redivivusmales are attracted to 1 mM and 1 μM of ascr#7, whereas C. elegans malesonly respond to 1 mM. This may help to create species-specific chemicalsignatures that are made up of different blends of ascarosides.

Example 5 Conclusions

Ascarosides could represent a universal nematode language, given theirwidespread production and recognition by free-living and parasiticnematodes. These findings are evocative of inter-species quorum sensingmolecules, Nacyl homoserine lactones (AHLs), which are both produced andsensed by many Gramnegative bacteria. AHLs are composed of the samehomoserine lactone but have species-specific variations in the N-acylchain40. Ascarosides are organized in a very similar fashion; they arecomposed of the same ascarylose sugar ring but have variations in theattached fatty acid tail. The inventors believe thissimilar-but-different organization of pheromones helps species tofacilitate both intra-specific and inter-specific communication. Thisdesign may also be useful to modify signals with limited cost, allowingnematodes to produce a variety of cues as necessary. A possiblemechanism of modifying preexisting ascarosides might help to producecommunicative signals quickly during times when swift response isnecessary.

For the same reason that there has been disagreement regarding theuncertainty of shared motives for quorum-sensing bacteria, there may notbe a unanimous motive for ascaroside production or recognition. It isclear that ascarosides are used for different reasons both between andwithin species, given that ascr#1 plays a small role in C. elegans dauerformation and since ascr#1 attracts P. redivivus males and repels P.redivivus females. Intraspecific discretion might be achieved by theproduction of a unique blend of ascarosides and/or the existence ofdifferent response thresholds. The findings support this possibility,given that nematodes produce different relative quantities ofascarosides and sometimes respond to different concentrations of thesame ascaroside. Because recombination is a theme often used in natureto achieve variation, it is not unlikely that nematodes secretecombinations from the same repertoire of ascarosides to present aspecific chemical signature to their surrounding environment. Once theyare secreted into the environment, ascarosides may potentially be usedby nematodes for any number of reasons, such as predation, food sharing,coordinated defense, cohabitation, diapause, mate-finding, aggregation,etc. Ascarosides mediate stress-resistance, mate finding, and clusteringbehavior. These survival strategies might be useful for the selectiveinhibition of a parasite's induction into its infectious stage, abilityto find mates, or their ability to mount a coordinated defense againstpredators. Alteration of the concentration and/or combination ofascarosides may also be useful to promote nematode survival, as thereare also many beneficial uses for nematodes. For example, nematodes fromthe genus Heterorhabditis are being used as a biological control agentfor the eradication of black vine weevils, citrus fruit weevils, fleas,wood boring leps, mushroom flies and many other agricultural pests.Lastly, the discovery of a common language between nematodes providesnew insight into mechanisms of animal communication, given that thediscovery of ascarosides as a shared nematode language represents a steptoward understanding small molecule signaling in this most abundantgroup of metazoans.

Example 6 Methods—Attraction Assay

OP50 E. coli is grown on a standard 5 cm agar plate (made with standardNematode Growth Medium). The bacterial lawn is 16 mm in diameter and isgrown overnight at 20 C before being used in trials. Two 4-mm spots (0.6L) were placed on opposite sides of the bacterial lawn (using atransparent template to guide spot placement) and several minuteselapsed for the liquid to settle in before placing nematodes down on theassay. Recording began immediately upon worm placement. 0.6 L of thecontrol was placed on one side of the lawn and 0.6 L of the experimentalcue was placed on the other side of the lawn, changing the location ofthe cue throughout trials, between left/right and top/bottom to avoidbias. Nematodes were isolated by gender at the L4 stage the day beforebeing used in trials as developed adults. Five worms were each placed attwo points equidistant from the foci of the scoring region (ten total).Trials were recorded for 20 minutes and frames were collected foranalysis at 1 frame per second. Results were averaged from at leastthree different trials. For every nematode species in this study, theinventors tested different total number of worms (using water in bothscoring regions) to determine the minimum number of worms necessary forconsistent unbiased results over a 20-minute trial. The total number ofworms used in the multiple species assays depending on that species'optimal parameters. 10 worms were for P. redivivus males and females, 20worms were used for C. elegans males, O. dolichuridae males, and C. sp7males and 14 worms were used for S. glaseri males.

Example 7 Methods—Automated Software

A video camera attached to the microscope produces a digital videostream, which is then analyzed. The ratio of time the average wormspends in each region of interest is calculated for every trial. Forease of implementation, it was assumed that all worms in a singleexperiment are roughly the same size. Thus, worm pixels were countedinstead of whole worms, allowing the inventors to take into accountfractions of a worm in the region of interest. It also eliminated theneed for a shape-based worm identification algorithm, and allowed eachframe to be analyzed independently. The inventors applied a band-passfilter to each frame to eliminate the effect of uneven lighting and alsoaccentuate the worms against the background. The worm was thenidentified after thresholding the filtered image. Throughout eachexperiment, the inventors know the locations and sizes of the regions ofinterest. Through the filtering described above, the inventors knowwhich pixels are occupied by worms and which ones are not. They are thenable to calculate the ratio of worm-pixels to all pixels inside theregion of interest to produce the worm-occupancy ratio. This is done forevery frame, giving a plot output of worm-occupancy ratio vs. time foreach region.

Example 8 Dauer Assay

The inventors adapted the dauer assay from Butcher et al. (2008) andused liquid cultures instead of agar plates. Nematode embryos weresynchronized by bleaching twice (3 hours apart) and collected in SComplete for liquid culture. They were grown in 4 mg/mL HB101 E. coli ata density of 6 worms per L, along with no ascarosides or 220 nM of theascaroside. They were incubated at 20 C for 4 days, after which theywere scored for % dauer formation using observation of anatomicalchanges and 2% SDS survival tests. Several hundred worms were scored foreach trial, with an average of at least three trials.

Example 9 Activity-Guided Fractionation

Activity-Guided Fractionation and NMR and HPLC-MS analysis of ascr#1from P. redivivus was similar to protocols developed for C. elegansmating pheromones.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of constituentmodules for the inventive compositions, and the diseases and otherclinical conditions that may be diagnosed, prognosed or treatedtherewith. Various embodiments of the invention can specifically includeor exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

The invention claimed is:
 1. A method of alleviating or treating aninflammatory disorder in a human subject, comprising: administering tothe subject a composition comprising isolated ascr#7

or a salt thereof; and wherein the inflammatory disorder is asthma,inflammatory bowel disease, or type one diabetes.
 2. The method of claim1, wherein the method reduces mucus production in the subject.
 3. Themethod of claim 1, wherein the naturally occurring ascaroside isproduced from a Nippostrongylus brasiliensis.
 4. The method of claim 1,wherein the inflammatory disorder is asthma.
 5. The method of claim 1,wherein the inflammatory disorder is inflammatory bowel disease.
 6. Themethod of claim 1, wherein the inflammatory disorder is type onediabetes.